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SAM 108.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 108

Supplemental Assay Method for the Detection of Extraneous Bovine Viral 

Diarrhea Virus in Modified-Live Vaccines

Date: October 16, 2014

Number: SAM 108.05

Supersedes: SAM 108.04, February 9, 2011

Standard Requirement: 9 CFR 113.300

Contact: Alethea M. Fry, (515) 337-7200

Peg A. Patterson

Approvals: /s/Geetha B. Srinivas Date: 31Dec14

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 06Jan15

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 09Jan15

Rebecca L.W. Hyde, Section Leader

Quality Management

Center for Veterinary Biologics

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA 

and does not imply its approval to the exclusion of other products that may be suitable.
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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagents/control procedures

3.4 Preparation of the sample

4. Performance of the Test

5. Interpretation of the Test Results

6. Report of Test Results

7. References

8. Summary of Revisions

Appendices
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1. Introduction

This Supplemental Assay Method (SAM) describes an in vitro test method using a cell culture 

procedure and a direct or an indirect fluorescent antibody technique (DFAT, IFAT) for detecting 

extraneous bovine viral diarrhea virus (BVDV) in modified-live vaccines (MLV) produced in 

cell cultures which support the growth of BVDV.

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1.1 Incubator, 36°± 2°C, high humidity, 5 ± 1% CO2

2.1.2 Microscope, inverted light 

2.1.3 Microscope, ultraviolet light (UV-light microscope)

2.1.4 Microscope slide, glass staining dish with rack (glass staining dish)

2.1.5 Laboratory stirrer/hot plate and magnetic stir bar

2.1.6 Centrifuge with rotor

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below.

2.2.1 Monospecific antiserum that is neutralizing, nontoxic to cell cultures, free 

of BVDV1 and BVDV2 antibodies, and specific to the agent(s) in the test serial.

2.2.2 Bovine turbinate (BT) secondary cell culture or other permissive cells free 

of extraneous agents as tested by title 9, Code of Federal Regulations, 

(9 CFR).

2.2.3 Minimum essential medium (MEM) (National Centers for Animal 

Health (NCAH) Media #20030)

1. 9.61 g MEM with Earles salts without bicarbonate
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2. 1.1 g sodium bicarbonate (NaHCO3)

3. Dissolve Steps 1 and 2 with 900 mL deionized water (DI).

4. Add 5 g lactalbumin hydrolysate or edamine to 10 mL DI. Heat to

60°± 2°C until dissolved. Add to Step 3 with constant stirring.

5. Q.S. to 1000 mL with DI; adjust pH to 6.8 to 6.9 with 2N hydrochloric 

acid (HCl).

6. Sterilize through a 0.22-μm filter.

7. Aseptically add:

a. 10 mL L-glutamine

b. 50 ug/mL gentamicin sulfate

8. Store at 2°- 7°C.

2.2.4 Growth Medium

1. 900 mL of MEM

2. Aseptically add 100 mL gamma-irradiated fetal bovine serum (FBS).

3. Store at 2°- 7°C.

2.2.5 Maintenance Medium

1. 980 mL of MEM

2. Aseptically add 20 mL gamma-irradiated fetal bovine serum (FBS).

3. Store at 2°- 7°C.

2.2.6 BVDV Reference Viruses

1. Noncytopathic BVDV type 1, e.g., New York-1 strain

2. Noncytopathic BVDV type 2, e.g., 890 strain
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3. Other BVDV strains (optional), e.g., Singer strain (cytopathic type 1), 

125 strain (cytopathic type 2)

2.2.7 For DFAT, anti-BVDV fluorescein isothiocyanate labeled polyclonal 

antiserum (anti-BVDV FITC conjugate), reactive to all type 1 and type 2 strains 

of BVDV.

2.2.8 For IFAT, a polyclonal antiserum reactive to all type 1 and type 2 strains 

of BVDV. For additional testing, BVD types 1 and 2 monoclonal antibodies 

(MAb) may also be used. 

2.2.9 For IFAT, appropriate anti-species IgG (H+L) fluorescein isothiocyanate 

labeled antiserum (anti-species FITC conjugate)

2.2.10 0.01 M Phosphate buffered saline (PBS) (NCAH Media #30054)

1. 1.19 g sodium phosphate, dibasic, anhydrous (Na2HPO4)

2. 0.22 g sodium phosphate, monobasic, monohydrate (NaH2PO4•H2O)

3. 8.5 g sodium chloride (NaCl)

4. Q.S. to 1000 mL with DI.

5. Adjust pH to 7.2 to 7.6 with 0.1 N sodium hydroxide (NaOH) or 2N 

HCl.

6. Sterilize by autoclaving at 15 psi, 121°± 2°C for 35 ± 5 minutes.

7. Store at 2°- 7°C.

2.2.11 Acetone

2.2.12 Cell culture flask, 25-cm

2

2.2.13 Cell culture slides, 8-chamber (Lab-Tek®slide)

2.2.14 Polystyrene tubes, 12 x 75-mm
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3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have experience in aseptic techniques, ultraviolet light microscopy, and 

cell culture techniques.

3.2 Preparation of equipment/instrumentation

On the day of the DFAT or IFAT, prepare a humidity chamber in the aerobic incubator 

by filling a pan in the bottom with DI.

3.3 Preparation of reagents/control procedures

3.3.1 One day prior to test initiation (first passage) and one day prior to second 

passage, seed one 25-cm

2

cell culture flask per test serial, one 25-cm

2

cell culture 

flask for a media control, and one 25-cm

2

cell culture flask for an uninoculated 

cell control, using 10 mL of approximately 105.2 to 105.5 BT cells/mL in Growth 

Medium. Incubate at 36°± 2°C in a CO2 incubator, to 50 to 75% confluency. 

These become the BT flasks.

3.3.2 One day prior to inoculation of the Lab-Tek®slides, seed one Lab-Tek®

slide per test serial with 0.3 to 0.4 mL of approximately 105.0to 106.5 BT cells/mL

in Growth Medium. Incubate at 36°± 2°C in a CO2 incubator, to 60 to 90% 

confluency in 24 hours.

3.3.3 Preparation of BVDV Positive Controls

On the day of inoculation of the Lab-Tek®slides, dilute the BVDV type 1 and 

type 2 reference viruses in MEM, according to the Center for Veterinary 

Biologics (CVB) Reagent Data Sheet or the manufacturer’s recommendations.

3.3.4 Preparation of working anti-BVDV FITC conjugate for DFAT

On the day of the DFAT, dilute anti-BVDV FITC conjugate in PBS to the 

working dilution according to the CVB Reagent Data Sheet or the manufacturer’s 

recommendations.
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3.3.5 Preparation of working polyclonal antiserum and BVDV type 1 and 2 

MAb for IFAT

On the day of the IFAT, dilute the polyclonal antiserum or BVDV types 1 and 2 

MAb in PBS to the IFA working dilution as determined for that specific 

antiserum or MAb according to the CVB Reagent Data Sheet or the 

manufacturer’s recommendations. The polyclonal antiserum should be used as a 

screening antibody, and the MAb may be used if further identification of 

genotype of virus is necessary.

3.3.6 Preparation of working anti-species FITC conjugate for IFAT

On the day of the IFAT, dilute the anti-species FITC conjugate in PBS to the 

working dilution according to the manufacturer’s recommendations.

3.4 Preparation of the sample

3.4.1 The initial test of a test serial will be with a single vial (a single sample 

from 1 vial).

3.4.2 On the day of test initiation, rehydrate the test serial according to the 

manufacturer’s instructions. If the test serial contains a bacterin for the diluent, 

sterile DI is used to replace the diluent. Mix by vortexing.

3.4.3 Test serials may require neutralization. (See Appendix II for a listing of 

viruses able to replicate in BT.)

1. Monovalent test serial: 0.5 mL of rehydrated test serial and 

0.7 mL of monospecific antiserum against virus able to replicate in BT 

that is a fraction in the test serial are mixed in a labeled 12 x 75-mm 

polystyrene tube. Depending on the titer of the virus to be neutralized and 

the antiserum neutralization titer, it may be necessary to increase the 

amount of antiserum used for neutralization of a given virus, as 

determined empirically. Incubate for 60 ± 10 minutes at room temperature.

2. Bivalent test serial: 0.5 mL of rehydrated test serial and 0.5 mL of 

each monospecific antisera against viruses able to replicate in BT that are 

a fraction in the test serial are mixed in a labeled 12 x 75-mm polystyrene 

tube. Depending on titer of the virus to be neutralized and the antiserum 

neutralization titer, it may be necessary to increase the amount of 

antiserum used for neutralization of a given virus, as determined 

empirically. Incubate for 60 ± 10 minutes at room temperature.
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3. Test serial containing 3 or more fractions: 0.5 mL of the rehydrated 

test serial and 0.3 mL of each monospecific antisera against viruses able to 

replicate in BT that are a fraction in the test serial are mixed in a labeled 

12 x 75-mm polystyrene tube. Depending on the titer of the virus to be 

neutralized and the antiserum neutralization titer, it may be necessary to 

increase the amount of antiserum used for neutralization of a given virus. 

Incubate for 60 ± 10 minutes at room temperature.

4. Performance of the Test

4.1 Just prior to test initiation, decant the Growth Medium from all but one of the BT 

flasks.

4.2 Inoculation

4.2.1 Inoculate 0.5 mL of test serial not requiring neutralization into a BT flask; 

q.s. to 2.5 mL with Maintenance Medium. This becomes the test flask.

4.2.2 When neutralization is required, inoculate the entire volume of the 

neutralized test serial (Section 3.4.3) after the incubation period into a BT flask,

q.s. to 2.5 mL with Maintenance Medium. This becomes the test flask.

4.3 Maintain 1 BT flask per test as an unopened, uninoculated cell control. 

4.4 Maintain 1 BT flask per test as a media control. Inoculate the media control with 

2.5 mL Maintenance Medium.

4.5 Allow each test flask and media control to absorb at 36°± 2°C for 60 ± 10 

minutes.

4.6 Add 7.5 mL Maintenance Medium to each test flask and the media control (i.e., 

q.s. to 10 mL).

4.7 Incubate the flasks at 36°± 2°C in a CO2 incubator. Observe each test flask, media 

control, and cell control not less often than every 3 days with an inverted light 

microscope for any signs of cytopathic effect (CPE) or contamination.

4.8 Four days ± 1 day postinoculation observe each test flask, media control, and cell 

control for any signs of CPE. Discard the cell control if no CPE is evident. Freeze the 

remaining flasks at -70°± 5°C for a minimum of 30 minutes. Flasks may be maintained at

-70°± 5°C until the next passage. Thaw each flask at room temperature and shake 
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contents. When the contents of the flask have thawed completely, pour into a sterile 12 x

75-mm plastic tube, and centrifuge at 1000 X g for 10 minutes. 

4.9 Decant Growth Medium from all but one of the new BT flasks seeded one day 

previously, as described in Section 3.3.1.

4.10 Pipette 1.0 mL of the supernatant fluid from the first passage (see Step 8) of a test 

serial into a new labeled BT flask from which the Growth Medium has been removed; 

q.s. to 2.5 mL with Maintenance Medium. Repeat for the media control. These are the 

new test flask and media control. A new BT flask is maintained as an unopened, 

uninoculated cell control. Pour off the remaining portion of the supernatant fluid into 

another sterile 12 x 75-mm tube and freeze at -70°C. 

4.10.1 Additional neutralizing antisera may be necessary to suppress replication 

of specific viral fractions in the vaccine. If so, the cell suspension neutralization 

may be performed according to Sections 3.4.3(1), 3.4.3(2), or 3.4.3(3).

4.10.2 Inoculate the entire volume of the neutralized cell suspension after the 

incubation period into a BT flask; q.s. to 2.5 mL with Maintenance Medium. This 

becomes the test flask.

4.11 Allow each test flask and media control to absorb at 36°± 2°C for 60 ± 10 

minutes, then q.s. to 10 mL with 7.5 mL Maintenance Medium, as in Steps 5 and 6.

4.12 Incubate flasks at 36°± 2°C in a CO2 incubator. Observe all flasks not less than 

every 3 days for signs of CPE or contamination.

4.13 Four days ± 1 day after the second inoculations repeat Step 8. This completes the 

second passage of the test serial.

4.14 Inoculate 0.1 mL/well of the cell suspension from each test flask into at least 4 

wells of a Lab-Tek®slide, seeded one day previously, as described in Section 3.3.2. For 

every assay, at least 4 wells serve as uninoculated cell control wells. Incubate at 36°± 2°C 

in a CO2 incubator for 4 days ± 1 day postinoculation.

4.14.1 Additional neutralizing antisera may be necessary to suppress replication 

of specific viral fractions in the vaccine. If so, add antiserum directly to the LabTek®

slide. 

4.14.2 Alternately, the cell suspension neutralization may be performed similar to 

Sections 3.4.3(1), 3.4.3(2), or 3.4.3(3). 
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4.15 Add 0.1 mL/well of each BVDV positive control to at least 4 wells of a Lab-Tek®

slide, seeded 1 day previously, as described in Section 3.3.2. Incubate at 36°± 2°C in a 

CO2 incubator for 4 days ± 1 day postinoculation.

4.16 Add 0.1 mL/well of the cell suspension from the media control flask to at least 4 

wells of a Lab-Tek®slide, seeded one day previously, as described in Section 3.3.2. 

Incubate at 36°± 2°C in a CO2 incubator for 4 days ± 1 day postinoculation.

4.17 Following incubation, decant the media from all Lab-Tek®slides. Remove the 

plastic walls by twisting them away from the Lab-Tek®slides, leaving the gasket 

attached.

4.18 Place the Lab-Tek®slides in a slide rack; place the rack in a glass staining dish 

filled with PBS. (Optional: A magnetic stir bar can be placed to rotate slowly in the dish, 

which is set on a laboratory stir plate.) Let stand 15 ± 5 minutes at room temperature. 

This step may be omitted in the event cells are in danger of sloughing from the Lab-Tek®

slides. In that case, cells are immediately fixed in acetone without a PBS rinse (see Step

19).

4.19 Discard the PBS, replace it with acetone, and fix the Lab-Tek®slides for 15 ± 5 

minutes at room temperature. Remove and allow to air dry.

4.20 Either DFAT or IFAT may be used to detect possible extraneous BVDV. A 

suitable anti-BVDV FITC conjugate used as a DFAT offers the advantage of less steps in 

processing the Lab-Tek®slides. For IFAT use polyclonal BVDV antiserum. If further 

characterization of the BVDV is required, it can be accomplished by using type 1- and 

type 2-specific antibodies provides information on the genotype of BVDV strains.

4.20.1 For DFAT, pipette 75 ± 25 μL of a working anti-BVDV FITC conjugate 

into each well of the Lab-Tek®slides. Incubate in a CO2 incubator at 36°± 2°C for 

30 ± 5 minutes.

4.20.2 For IFAT, pipette 75 ± 25 μL of a working polyclonal BVDV antiserum 

into at least 4 wells of a Lab-Tek®slide inoculated with a given test serial. If 

desired, pipette 75 ± 25 μL of a working BVDV type 1 MAb or type 2 MAb into 

at least 4 wells each of slide inoculated with the same test serial. Repeat this for 

every test serial. Pipette 75 ± 25 μL of each working BVDV polyclonal antiserum 

and MAb into at least 4 wells of cell control, and at least 4 wells of media control. 

Incubate in a CO2 incubator at 36°± 2°C for 30 ± 5 minutes.

1. Wash per Step 18. Discard the PBS.
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2. Pipette 75 ± 25 μL of the working anti-species FITC conjugate into 

each well of the Lab-Tek®slides; incubate in a CO2 incubator at 36°± 2°C 

for 30 ± 5 minutes.

4.21 Wash per Step 18. Discard the PBS. Briefly dip each Lab-Tek®slide in DI.

4.22 Allow the Lab-Tek®slides to air dry.

4.23 Read within 1 hour at 100-200X with a UV-light microscope. Alternatively, the 

Lab-Tek®slides may be stored in the dark at 2°- 7°C for no longer than 48 hours before 

reading. Examine the cell monolayer for typical apple-green cytoplasmic fluorescence.

4.24 Wells containing 1 or more cells displaying specific fluorescence for BVDV are 

positive.

5. Interpretation of the Test Results

5.1 Validity requirements

5.1.1 For a valid test, the media control and the cell control wells on Lab-Tek®

slides shall not exhibit signs of contamination, CPE, or positive DFAT or IFAT.

5.1.2 The BVDV positive controls must show specific fluorescence for BVDV.

5.1.3 If either validity requirement in Sections 5.1.1 or 5.1.2 is not met, the test 

is considered a NO TEST and may be repeated without prejudice.

5.2 Results

5.2.1 If the initial test is valid and all wells of the test serial on the Lab-Tek®

slides are free of BVDV contamination, the serial is SATISFACTORY.

5.2.2 Retests

1. If the initial test is valid and 1 or more wells of the test serial on the 

Lab-Tek®slides exhibits signs of BVDV contamination, the test is 

repeated (1st retest), using a new vial of the test serial. Retests are 

conducted using an additional BT flask for each passage and at least 4 

wells of a Lab-Tek®slide to serve as serum control flask and wells, 

respectively. These will have contents identical to the media control flask 

and wells, plus addition of equivalent amounts of antiserum as used in the 

test flask and wells to neutralize viral fractions of the test serial. 
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2. If the second valid test (1st retest) confirms the initial result and the 

serum control is negative for BVDV, the test serial is 

UNSATISFACTORY.

3. If the second valid test (1st retest) fails to confirm the initial result, the 

test serial is tested a third time (2nd retest). The test is repeated using a new 

vial of test serial and a serum control as described in Step 1.

a. If the second and third valid tests (1st and 2nd retests) result in no 

signs of BVDV contamination, the test serial is SATISFACTORY.

b. If the third valid test (2nd retest) confirms the initial result and the 

serum control is negative for BVDV, the test serial is 

UNSATISFACTORY.

4. If at any time in a valid retest the serum control in the flasks or on the 

Lab-Tek®slides is positive for BVDV, the blocking antiserum used in the 

testing is suspect and that test and all preceding testing is a NO TEST. A 

test serial may be retested without prejudice after adequate evaluation and 

assurance of BVDV free reagents.

6. Report of Test Results

Record all test results on the test record.

7. References

7.1 Title 9, Code of Federal Regulations, part 113.300, U.S. Government Printing 

Office, Washington, DC.

7.2 G. E. Cottral ed. Manual of Standardized Methods for Veterinary Microbiology. 

Comstock Publishing Assoc. Ithaca, NY.

8. Summary of Revisions

Version .05

 The Contact information has been updated; however, the Virology Section has elected 

to keep the same next review date for the document.
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Version .04

 The Contact information has been updated.

 3.3.2: Revised section to include a larger range of cell density and confluency for 

seeded :LabTek slides. 

Version .03

 The Contact has been changed from Kenneth Eernisse and Marsha Hegland to Joseph 

Hermann and Peg Patterson.

 2.1: The 36° aerobic incubator has been deleted and a centrifuge and rotor have been 

added.

 4.20: The use of an aerobic incubator has been changed to a CO2 incubator.

Version .02

This document was revised to clarify practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail. While no significant changes were made that impact 

the outcome of the test, the following changes were made to the document: 

 2.2.3 The forumlation of MEM has been changed to include 1.1 grams sodium 

bicarbonate per liter instead of 2.2 grames, and the removal of penicillin, 

streptomycin, and amphotericin B.

 The refrigeration temperatures have been changed from 4° ± 2°C to 2°- 7°C

throughout the document. 
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Appendix I

Cell lines that support BVDV replication

Based on testing conducted at the CVB and other scientific institutions, the cell lines that 

are permissive for BVDV include:

1) all bovine, ovine, and swine cell lines,

2) cat cell lines, especially Crandell feline kidney (CRFK),

3) rabbit cell lines,

4) MA104 (African green monkey) cell lines,

5) chick embryo fibroblast cells and cell lines,

6) equine dermal cell lines (ED), though the BVDV titer is much reduced.

Products grown on cells that are not readily available to the CVB, such as ferret embryo 

cells, are also considered candidates for testing until such time as BVDV replication on 

these cells is determined.
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Appendix II

Agents that require neutralization during testing

Live vaccine viruses or live agents that replicate when grown in BT and that may 

potentially interfere with the detection of extraneous BVDV are neutralized with 

homologous antisera. 

Agents requiring neutralization Agents not requiring neutralization

bovine coronavirus

bovine parvovirus

bovine rotavirus 

bovine respiratory syncytial virus

bluetongue virus 

Chlamydia psittaci*

canary pox virus

canine distemper virus

canine parainfluenza virus

equine herpesvirus 1 and 4 

equine influenza virus

infectious bovine rhinotracheitis virus

infectious canine hepatitis virus

measles virus

parainfluenza virus 3

pseudorabies virus

canine coronavirus

canine parvovirus

equine viral arteritis virus

feline calici virus

feline infectious peritonitis virus

feline panleukopenia virus

feline rhinotracheitis virus

porcine parvovirus

West Nile virus

* When present in viral vaccines, Chlamydia psittaci may cause unwanted effects on BT. 

These effects can be neutralized by the use of tetracycline.